Electrophoresis
A common method of analyzing nucleic acid (e.g., DNA or RNA) fragments is by separation in an agarose or polyacrylamide gel matrix. Such matrices provide a medium in which such fragments of different sizes and forms may be analyzed. In the presence of an electrical current, nucleic acid fragments will migrate through such a gel matrix in the direction of the positive electrode. Since small, linear nucleic acid molecules migrate more easily and quickly through the pores of the gel than do larger molecules, the matrix acts as a molecular sieve, separating fragments of different sizes. Larger nucleic acid fragments are typically electrophoresed on low concentration agarose gels, while smaller fragments are separated on higher concentration agarose gels or on polyacrylamide gels, since polyacrylamide has a higher resolution capacity than agarose.
Detection of Nucleic Acid Fragments
Detection of nucleic acid molecules electrophoresed in agarose or polyacrylamide gels can be accomplished by a variety of techniques, including the use of fluorescent dyes such as ethidium bromide and SYBR Green. These dyes bind to the nucleic acid molecules and fluoresce when exposed to UV light. Alternatively, the nucleic acid molecules can be detectably labeled by chemically coupling them with radioactive, fluorescent or chemiluminescent labels.
Sizing Standards
For the purpose of analyzing nucleic acid fragments on agarose or polyacrylamide gels, nucleic acid molecular weight standards are very useful tools, providing the researcher with a point of reference for estimating the quality, size, and/or quantity of the nucleic acid sample. A standard is typically run simultaneously on the gel with the sample (e.g., in parallel with the sample), and following detection, a comparison is made between the sample band(s) and the bands of the standard. Knowing the size (in base pairs) of the standard allows the size of the unknown fragment(s) to be estimated.
Common standards used for estimating the size of nucleic acid fragments on gels include naturally occurring genomic DNA of bacteriophages (e.g., lambda bacteriophage and φX174). To prepare such standards, the phage genomic DNA is cleaved into a population of nucleic acid fragments of known size using a specific restriction endonuclease. These types of standards are commonly called “nucleic acid markers” (e.g., “DNA markers”).
Another type of nucleic acid standard is produced by engineering plasmids to contain recognition/cleavage sites for one or more specific restriction endonucleases at particular intervals in the plasmid. See, e.g., WO 95/11971. Upon digestion of the plasmid with the specific endonuclease(s), nucleic acid fragments of specific known sizes are generated. For accuracy in size determination and for ease of use, it is beneficial to have numerous bands that increase in size in regular, even intervals. The bands of the standard should also be able to be detected with equivalent intensity. These types of standards are commonly called “nucleic acid ladders” (e.g., “DNA ladders”).
A 10 bp DNA ladder which consists of a set of twenty fragments containing repeats of a 10 bp sequence is commercially available from Life Technologies, Inc. (Gaithersburg, Md.). This ladder is constructed by ligation of synthetic double-stranded oligonucleotides, and is not generated from a plasmid. The multimers in the ladder contain multiple deoxyuracil bases in one strand and can be converted to a single-stranded form by incubation with uracil DNA glycosylase (UDG), which degrades the uracil-containing strand. Bands at 10 bp and 100 bp are highlighted for orientation within the standard.
This 10 bp DNA ladder can be used for sizing double-stranded DNA, or treated with UDG as described above for sizing single-stranded DNA, and may be stained with ethidium bromide or end-labeled with T4 polynucleotide kinase. However, due to the nature of the production procedure (ligation of oligonucleotides), it is not amenable to fluorescent detection, nor is it useful for Amplification Fragment Length Polymorphism (AFLP) analysis (Vos et al., Nucl. Acids. Res. 23:4407-4414 (1995)), primarily because the resulting range of fragment sizes is too low and the electrophoretic bands are not sharply resolved.
Other commercially available products include a 20 bp DNA ladder sold by GenSura, a 20 bp DNA ladder sold by Invitrogen (these may be the same product sold by the two companies), and LTI's 25 bp ladder. The LTI 25 bp DNA ladder is derived from a plasmid engineered to contain several repeats of a 25 bp sequence which can be digested by restriction enzymes to generate bands increasing in size by 25 bp. The repeated sequence in the LTI 25 bp DNA ladder, however, can be used only for sizing double-stranded DNA.